Bacterially derived intact minicells that encompass plasmid free functional nucleic acid for in vivo delivery to mammalian cells

ABSTRACT

Intact, bacterially-derived minicells can safely introduce therapeutically effective amounts of plasmid-free functional nucleic acid to target mammalian cells. To this end, functional nucleic acid can be packaged into intact minicells directly, without resort to expression constructs, the expression machinery of the host cell, harsh chemicals or electroporation.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/980,781, filed Dec. 29, 2010, now U.S. Pat. No. 8,669,101, which is adivisional of U.S. patent application Ser. No. 12/053,197, filed Mar.21, 2008, now U.S. Pat. No. 8,735,566, which claims priority from U.S.Provisional Patent Application No. 60/909,074, filed Mar. 30, 2007.Also, U.S. patent application Ser. No. 12/053,197 is acontinuation-in-part of U.S. patent application Ser. No. 11/211,098,filed Aug. 25, 2005, now U.S. Pat. No. 8,691,963, which claims priorityfrom U.S. Provisional Patent Application No. 60/604,433, filed Aug. 26,2004. The respective contents of these applications are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

Recently, a number of nucleic acid-based strategies have been developedto modulate a variety of cellular functions (Opalinska and Gewirtz,2002). Classes of oligonucleotides such as aptamers, transcriptionfactor-binding decoy oligonucleotides, ribozymes, triplex-formingoligonucleotides, immunostimulatory CpG motifs, antisenseoligonucleotides (including peptide nucleic acids), small interferingRNAs, and microRNAs have drawn much interest as research tools, owing totheir highly specific mode of action. These oligomeric nucleic acidshave considerable potential as therapeutics, too. Such therapeutics faceseveral obstacles, however, including the instability of free nucleicacids and the safe, efficient and targeted cellular delivery of thesemacromolecules (Dykxhoorn and Lieberman, 2005).

A focus of many nucleic acid-based therapeutic strategies is thephenomenon of RNA interference (RNAi), whereby long, double-stranded RNA(dsRNA) in a cell leads to sequence-specific degradation of homologous(complementary or partially complementary) gene transcripts. Moreparticularly, the long dsRNA molecules are processed into smaller RNAsby an endogenous ribonuclease called “Dicer” (Grishok et al., 2000;Zamore et al., 2000). The smaller RNAs are known as “short interferingRNA” (siRNA) when they derive from exogenous sources and as “microRNA”(miRNA) when they are produced from RNA-coding genes in the cell's owngenome. These two classes of small (typically, 21- to 23-nucleotide)regulatory RNAs also differ in that miRNAs show only partialcomplementarity to messenger RNA (mRNA) targets.

The short regulatory RNAs bind to the so-called “RNA-induced silencingcomplex” (RISC), which has a helicase activity and an endonucleaseactivity. The helicase activity unwinds the two strands of RNAmolecules, allowing the antisense strand to bind to the targeted RNAmolecule (Zamore et al., 2000; Zamore, 2002; Vickers et al., 2003). Theendonuclease activity hydrolyzes the target RNA at the site where theantisense strand is bound.

In RNAi, therefore, a single-stranded RNA molecule (ssRNA) binds to thetarget RNA molecule by Watson-Crick base-pairing rules and recruits aribonuclease that degrades the target RNA. By contrast, antisensesuppression of gene expression entails the binding of ssRNA to mRNA,blocking translation without catalyzing the degradation of the mRNA.

As a class, regulatory RNAs have a half-life of less than an hour inhuman plasma (Layzer et al., 2004) and are rapidly excreted by thekidneys. Consequently, several groups have attempted to prepareregulatory RNAs, including siRNAs, that are nuclease-resistant. Examplesof such efforts include chemically modifying the nucleotides (e.g.,2′-F, 2′-OMe, Locked Nucleic Acids; LNA) or the phosphodiester backbone,e.g., phosphorothioate linkages (Chiu and Rana 2003; Choung et al.,2006; Czauderna et al. 2003; Elmén et al., 2005; Layzer et al., 2004;Morrissey et al., 2005). Also, to minimize the time siRNAs or otherregulatory RNAs spend in circulation, practitioners have conjugated theRNA molecules to proteins and antibodies to target desired mammaliancells. In further efforts to address the concerns of low stability andrapid renal excretion, practitioners have developed vehicles fordelivering regulatory RNAs. Polyplexes (formed by self assembly ofnucleic acids with polycations), lipopolyplexes (formed by initialcondensation of the nucleic acid with polycations, followed by additionof cationic lipids), liposomes, and synthetic nanoparticles are beingexplored, too.

These approaches also face numerous obstacles, such as (a) rapidclearance of the carrier proteins from the serum through renalexcretion, (b) limited number of regulatory RNA molecules that can beconjugated to each carrier protein, (c) difficulty in intracellulardissociation of intact, regulatory RNAs from the carrier protein, (d)rapid clearance due to polyplexes binding serum proteins which can actas opsonins (Dash et al., 1999), and (e) instability of liposomes invivo, causing release of nucleic acids into the serum and potentialnon-specific transformation.

Viral vectors also have been developed to produce regulatory RNAsendogenously. See, e.g., Devroe and Silver, 2004. These viral vectorspose serious safety concerns, however. Illustrative problems includerecombination with wild-type viruses, insertional and oncogenicpotential, virus-induced immunosuppression, limited capacity of theviral vectors to carry large segments of DNA, reversion to virulence ofattenuated viruses, difficulties in manufacture and distribution, lowstability, and adverse reactions (Hacein-Bey-Abina et al., 2003;Kootstra and Verma, 2003; Raper et al., 2003; Verma and Weitzman, 2005;Check, 2005).

Plasmid-based systems also have been developed for recombinant, in situexpression of a regulatory RNA, such as an siRNA or a larger (˜70 nt)precursor, a short hairpin RNA (shRNA). An shRNA contains sense andantisense sequences from a target gene that are connected by a hairpinloop. See, e.g., Paddison et al., 2002. shRNAs can be expressed from apol-III-type promoter or, in the context of a miRNA, by pol IIpromoters.

As described in international application WO 03/033519, plasmids thatcode for an shRNA, siRNA, or other regulatory RNA can be transformedinto a parent bacterial strain that produces intact minicells, by virtueof a mutation that causes asymmetric cell division. Such transformationyields recombinant bacteria in which the plasmid replicatesintracellularly, introducing large numbers of plasmids in the bacterialcytoplasm. During the asymmetric division, some of the plasmidssegregate into the minicell cytoplasm, resulting in recombinantminicells. The minicells then can deliver the plasmid DNA into amammalian cell, where the plasmid DNA migrates to the cell nucleus. Inthe nucleus the plasmid DNA expresses the shRNA or other regulatory RNA,as the case may be, and the resultant nucleic acid then migrates to thecytoplasm, where it can effect RNAi or gene suppression, depending onthe nature of the involved regulatory RNA.

Because such approaches require host machinery, however, deliveringtherapeutically effective amounts of nucleic acid via expression-basedsystems involves complex and protracted processes, which limits theireffectiveness. Accordingly, a more efficacious methodology is needed fordelivering functional nucleic acids, such as regulatory RNAs, to targetcells.

SUMMARY OF THE INVENTION

In accordance with one aspect of the invention, therefore, a compositioncomprising (a) a plurality of intact minicells, each minicell of theplurality encompassing plasmid-free functional nucleic acid, and (b) apharmaceutically acceptable carrier therefor. The category of“functional nucleic acid” is illustrated by single-, double-, ormulti-stranded DNA or RNA. In one embodiment, minicells of the pluralitycontain plasmid-free functional nucleic acid that is regulatory RNA.Examples of such regulatory RNA include, but are not limited to, siRNA,miRNA, and shRNA.

The minicell-packaged functional nucleic acid may target the RNAtranscripts encoding a protein that contributes to drug resistance, toapoptosis resistance, or to neoplasticity, inter alia. Also, acomposition of the invention may further comprise a bispecific ligandcomprised, for example, of a first arm specific for a minicell surfacestructure and a second arm specific for a non-phagocytic mammalian cellsurface receptor.

In another aspect of the invention, a method is provided for deliveringa functional nucleic acid to a target mammalian cell. The inventivemethodology comprises (a) providing a plurality of intact minicells in apharmaceutically acceptable carrier, each minicell of the pluralityencompassing plasmid-free functional nucleic acid, and (b) bringingminicells of the plurality into contact with mammalian cells such thatthe mammalian cells engulf minicells of the plurality, whereby thefunctional nucleic acid is released into the cytoplasm of the targetcells. As mentioned, the functional nucleic acid, exemplified byregulatory RNA such as siRNA, miRNA and shRNA, can target RNAtranscripts encoding a protein that contributes to drug resistance,apoptosis resistance or neoplasticity. In other embodiments, themethodology of the invention further comprises delivering a drug,distinct from the functional nucleic acid, to the target mammalian cell.The drug can be administered after or concurrently or even before theadministration of the minicell composition.

In accordance with another aspect, the present invention contemplates amethod for formulating a minicell with a plasmid-free functional nucleicacid. The method comprises co-incubating a plurality of minicells with afunctional nucleic acid, such as regulatory RNA like siRNA, miRNA orshRNA, in a buffer. In some embodiments, the co-incubation may involvegentle shaking, while in others the co-incubation is static. In someaspects, the co-incubation lasts about half an hour, while in others itlasts about an hour. In one embodiment, the buffer comprises bufferedsaline, for example, a 1× phosphate buffer solution. In anotherembodiment, the co-incubation is conducted at a temperature of about 4°C. to about 37° C., about 20° C. to about 30° C., about 25° C., or about37° C. The co-incubation can comprise about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹,10¹² or 10¹³ minicells.

Other objects, features and advantages will become apparent from thefollowing detailed description. The detailed description and specificexamples are given for illustration only since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.Further, the examples demonstrate the principle of the invention andcannot be expected to specifically illustrate the application of thisinvention to all the examples where it will be obviously useful to thoseskilled in the prior art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts intact minicells packaged with Cy3 fluorophore-labeledsiRNA. FIG. 1A is from a light microscope, while FIG. 1B shows the sameslide but viewed under fluorescent light with a 515-560 excitationfilter revealing strongly fluorescent siRNA molecules coincident withthe minicells.

FIG. 2 is an image captured by fluorescence confocal microscopy andshows the adhesion and internalization of EGFR-targeted,siRNA-Plk1-packaged minicells into human breast cancer cells in-vitro.

FIG. 3 graphically shows a significant anti-tumor effect achieved bytreating human breast cancer (MDA-MB-468) xenografts in nude mice withEGFR-targeted, KSP-siRNA-packaged minicells. Control group 1(

) received sterile saline, while experimental group 2 (

) received ^(EGFR)minicells_(siRNA-KSP). (10⁹), four times per week.

FIG. 4 graphically depicts a significant anti-tumor effect achieved bytreating human colon cancer (HCT116) xenografts in nude mice withEGFR-targeted, KSP-siRNA-packaged minicells in conjunction withEGFR-targeted, carboplatin-packaged minicells. Group 1 (

) mice received sterile saline, and mice of groups 2 (

), 3 (

) and 4 (

) were treated for the first 10 doses (see FIG. 4) with10^(9 EGFR)minicells_(siRNA-Plk1), ^(EGFR)minicells_(siRNA-KSP-1), and^(EGFR)minicells_(siRNA-KSP-2), respectively.

FIG. 5 provides FACS analysis, from various times post-transfection, ofcolon cancer (HCT116) cells treated with experimental minicells,^(EGFR)minicells_(siRNA-KSP), or ^(EGFR)minicells_(siRNA-Plk1). FIGS.5A-5D provide FACS analysis of samples harvested 4 hours aftertransfection, while FIGS. 5E-5H show analysis from samples at 8 hourspost-transfection. FIGS. 5A & 5E show results from cells only, whileFIGS. 5B & 5F concern cells+empty ^(EGFR)minicells. FIGS. 5C & 5G showresults from cells+^(EGFR)minicells_(siRNA-KSP), and FIGS. 5D & 5Hconcern cells+^(EGFR)minicells_(siRNA-Plk1).

FIG. 6 provides FACS analysis, from various times post-transfection, ofcolon cancer (HCT116) cells treated with experimental minicells,^(EGFR)minicells_(siRNA-KSP), or ^(EGFR)minicells_(siRNA-Plk). FIGS.6A-6D provide FACS analysis of samples harvested 16 hours aftertransfection, while FIGS. 6E-6H show analysis from samples at 24 hourspost-transfection. FIGS. 6A & 6E show results from cells only, whileFIGS. 6B & 6F concern cells+empty ^(EGFR)minicells. FIGS. 6C & 6G showresults from cells+^(EGFR)minicells_(siRNA-KSP), and FIGS. 6D & 6Hconcern cells+^(EGFR)minicells_(siRNA-Plk1).

FIG. 7 provides FACS analysis, from various times post-transfection, ofcolon cancer (HCT116) cells treated with experimental minicells,^(EGFR)minicells_(siRNA-KSP), or ^(EGFR)minicells_(siRNA-Plk1). FIGS.7A-7D provide FACS analysis of samples harvested 32 hours aftertransfection, while FIGS. 7E-7H show analysis from samples at 48 hourspost-transfection. FIGS. 7A & 7E show results from cells only, whileFIGS. 7B & 7F concern cells+empty ^(EGFR)minicells. FIGS. 7C & 7G showresults from cells+^(EGFR)minicells_(siRNA-KSP), and FIGS. 7D & 7Hconcern cells+^(EGFR)minicells_(siRNA-Plk1).

DETAILED DESCRIPTION OF THE INVENTION

Pursuant to the present invention, therapeutically effective amounts offunctional nucleic acid can be packaged into minicells without resort toharsh chemicals or electroporation. In this regard, a simple methodologyfor directly packaging such therapeutically effective concentrations offunctional nucleic acids into intact minicells has been developed thatdoes not involve plasmid-based expression constructs or the expressionmachinery of a host bacterial cell. Accordingly, a polynucleotidesegment that codes for the functional nucleic acid is not cloned into aplasmid DNA or viral vector. Instead, the plasmid-free functionalnucleic acids are packaged directly into the minicells by passingthrough the minicell's intact membrane. Moreover, a minicell compositionof the invention safely and effectively can deliver, to targetedmammalian cells, therapeutically effective amounts of functional nucleicacid molecules, illustrated by regulatory RNAs such as siRNAs, miRNAs,and shRNAs.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used inthis description have the same meaning as commonly understood by thoseskilled in the relevant art.

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.Other terms and phrases are defined throughout the specification.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

“Antisense oligonucleotide” refers to a nucleic acid moleculecomplementary to a portion of a particular gene transcript that canhybridize to the transcript and block its translation. An antisenseoligonucleotide can comprise RNA or DNA.

“Biomolecular sequence” or “sequence” refers to all or a portion of apolynucleotide or polypeptide sequence.

“Cancer,” “neoplasm,” “tumor,” “malignancy” and “carcinoma,” usedinterchangeably herein, refer to cells or tissues that exhibit anaberrant growth phenotype characterized by a significant loss of controlof cell proliferation. The methods and compositions of this inventionparticularly apply to malignant, pre-metastatic, metastatic, andnon-metastatic cells.

“Complementary” refers to the topological compatibility or matchingtogether of the interacting surfaces of two molecules, such as a siRNAmolecule and its target mRNA. The molecules can be described ascomplementary, and furthermore, the contact surface characteristics arecomplementary to each other.

“Corresponds to” or “represents” when used in the context of, forexample, a polynucleotide or sequence that “corresponds to” or“represents” a gene means that a sequence of the polynucleotide ispresent in the gene or in the nucleic acid gene product, e.g., mRNA. Thepolynucleotide can be wholly present within an exon of a genomicsequence of the gene, or different portions of the sequence of thepolynucleotide can be present in different exons, e.g., such that thecontiguous polynucleotide sequence is present in an mRNA, either pre- orpost-splicing, that is an expression product of the gene.

A “decoy RNA” is a molecule which can adopt a structure identical to animportant functional region of the RNA to be targeted. The latter RNAcan be native to a mammalian host or a pathogen that has infected amammalian cell, e.g. HIV. The decoy RNA sequesters away the protein thatnormally interacts with the target RNA resulting in a disruption ofnormal processing of the mammalian or pathogen host.

“Drug” refers to any physiologically or pharmacologically activesubstance that produces a local or systemic effect in animals,particularly mammals and humans.

“Expression” generally refers to the process by which a polynucleotidesequence undergoes successful transcription and translation such thatdetectable levels of the amino acid sequence or protein are expressed.In certain contexts herein, expression refers to the production of mRNA.In other contexts, expression refers to the production of protein.

“Functional nucleic acid” refers to a nucleic acid molecule that, uponintroduction into a host cell, specifically interferes with expressionof a protein. In general, functional nucleic acid molecules have thecapacity to reduce expression of a protein by directly interacting witha transcript that encodes the protein. Regulatory RNA, such as siRNA,shRNA, short RNAs (typically less than 400 bases in length), micro-RNAs(miRNAs), ribozymes and decoy RNA, and antisense nucleic acidsconstitute exemplary functional nucleic acids.

“Gene” refers to a polynucleotide sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full length codingsequence or by any portion of the coding sequence. A gene can constitutean uninterrupted coding sequence or it can include one or more introns,bound by the appropriate splice junctions. Moreover, a gene can containone or more modifications in either the coding or the untranslatedregions that could affect the biological activity or the chemicalstructure of the expression product, the rate of expression, or themanner of expression control. Such modifications include, but are notlimited to, mutations, insertions, deletions, and substitutions of oneor more nucleotides. In this regard, such modified genes can be referredto as “variants” of the “native” gene.

“Host cell” refers to a cell that can be, or has been, used as arecipient for a recombinant plasmid or other transfer ofpolynucleotides, and includes the progeny of the original cell that hasbeen transfected. The progeny of a single cell can not necessarily becompletely identical in morphology or in genomic or total DNA complementas the original parent due to natural, accidental, or deliberatemutation.

“Hybridization” refers to any process by which a polynucleotide sequencebinds to a complementary sequence through base pairing.

“Individual,” “subject,” “host,” and “patient,” used interchangeably inthis description, refer to any mammalian subject for whom diagnosis,treatment, or therapy is desired. In one preferred embodiment, theindividual, subject, host, or patient is a human. Other subjects caninclude but are not limited to cattle, horses, dogs, cats, guinea pigs,rabbits, rats, primates and mice.

“Label” refers to agents that are capable of providing a detectablesignal either directly or through interaction with one or moreadditional members of a signal producing system. Labels that aredirectly detectable and can find use in the invention includefluorescent labels. Specific fluorophores include fluorescein,rhodamine, BODIPY, cyanine dyes and the like. The invention alsocontemplates the use of radioactive isotopes, such as ³⁵S, ³²P, ³H, andthe like as labels. Colorimetric labels such as colloidal gold orcolored glass or plastic (e.g., polystyrene, polypropylene, latex) beadscan also be utilized. For instance, see U.S. Pat. No. 4,366,241, U.S.Pat. No. 4,277,437, U.S. Pat. No. 4,275,149, U.S. Pat. No. 3,996,345,U.S. Pat. No. 3,939,350, U.S. Pat. No. 3,850,752, and U.S. Pat. No.3,817,837.

“Oligonucleotide” refers to a polynucleotide comprising, for example,from about 10 nucleotides (nt) to about 1000 nt. Oligonucleotides foruse in the invention are preferably from about 10 nt to about 150 nt.The oligonucleotide can be a naturally occurring oligonucleotide or asynthetic oligonucleotide. Oligonucleotides can be modified.

“Minicell” refers to anucleate forms of bacterial cells, engendered by adisturbance in the coordination, during binary fission, of cell divisionwith DNA segregation. Minicells are distinct from other small vesiclesthat are generated and released spontaneously in certain situations andare not due to specific genetic rearrangements or episomal geneexpression. In the context of this invention the minicells are intactsince other “denuded” forms, such as spheroplasts, poroplasts,protoplasts, would leak the packaged functional nucleic acid and wouldnot be therapeutically effective. The intact minicell membrane allowsthe payload to be retained within the minicell and is releasedintracellularly within the target host mammalian cell.

In this description, “modified” and “chemically modified” refer tooligonucleotides or polynucleotides with one or more chemical changes tothe natural molecular structures of all or any of the bases, sugarmoieties, and internucleoside phosphate linkages, as well as tomolecules having added substitutions or a combination of modificationsat these sites. The internucleoside phosphate linkages can bephosphodiester, phosphotriester, phosphoramidate, siloxane, carbonate,carboxymethylester, acetamidate, carbamate, thioether, bridgedphosphoramidate, bridged methylene phosphonate, phosphorothioate,methylphosphonate, phosphorodithioate, bridged phosphorothioate orsulfone internucleotide linkages, or 3′-3′, 5′-3′, or 5′-5′ linkages,and combinations of such similar linkages. The phosphodiester linkagecan be replaced with a substitute linkage, such as phosphorothioate,methylamino, methylphosphonate, phosphoramidate, and guanidine, and theribose subunit of the polynucleotides also can be substituted (e.g.,hexose phosphodiester; peptide nucleic acids). The modifications can beinternal (single or repeated) or at the end(s) of the oligonucleotidemolecule, and can include additions to the molecule of theinternucleoside phosphate linkages, such as deoxyribose and phosphatemodifications which cleave or crosslink to the opposite chains or toassociated enzymes or other proteins. The terms “modifiedoligonucleotides” and “modified polynucleotides” also includeoligonucleotides or polynucleotides comprising modifications to thesugar moieties (e.g., 3′-substituted ribonucleotides ordeoxyribonucleotide monomers), any of which are bound together via 5′ to3′ linkages.

The phrase “nucleic acid molecules” and the term “polynucleotides”denote polymeric forms of nucleotides of any length, eitherribonucleotides or deoxynucleotides. They include single-, double-, ormulti-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or apolymer comprising purine and pyrimidine bases or other natural,chemically or biochemically modified, non-natural, or derivatizednucleotide bases. The backbone of a polynucleotide can comprise sugarsand phosphate groups (as can typically be found in RNA or DNA), ormodified or substituted sugar or phosphate groups. Alternatively, thebackbone of the polynucleotide can comprise a polymer of syntheticsubunits such as phosphoramidites and thus can be anoligodeoxynucleoside phosphoramidate or a mixedphosphoramidate-phosphodiester oligomer. A polynucleotide can comprisemodified nucleotides, such as methylated nucleotides and nucleotideanalogs, uracyl, other sugars, and linking groups such as fluororiboseand thioate, and nucleotide branches. A polynucleotide can be modifiedfurther, such as by conjugation with a labeling component. Other typesof modifications include caps, substitution of one or more of thenaturally occurring nucleotides with an analog, and introduction ofmeans for attaching the polynucleotide to proteins, metal ions, labelingcomponents, other polynucleotides, or a solid support.

“Pharmaceutically acceptable” refers to physiological compatibility. Apharmaceutically acceptable carrier or excipient does not abrogatebiological activity of the composition being administered, is chemicallyinert and is not toxic to the organism in which it is administered.

The qualifier “plasmid-free” connotes the absence of a construct, suchas a plasmid or viral vector, for in situ expression of a functionalnucleic acid.

“Polypeptide” and “protein,” used interchangeably herein, refer to apolymeric form of amino acids of any length, which can includetranslated, untranslated, chemically modified, biochemically modified,and derivatized amino acids. A polypeptide or protein can be naturallyoccurring, recombinant, or synthetic, or any combination of these.Moreover, a polypeptide or protein can comprise a fragment of anaturally occurring protein or peptide. A polypeptide or protein can bea single molecule or can be a multi-molecular complex. In addition, suchpolypeptides or proteins can have modified peptide backbones. The termsinclude fusion proteins, including fusion proteins with a heterologousamino acid sequence, fusions with heterologous and homologous leadersequences, with or without N-terminal methionine residues,immunologically tagged proteins, and the like.

“Purified” refers to a compound that is removed from its naturalenvironment and is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% free fromother components with which it is naturally associated.

“Regulatory RNA” denotes a category inclusive of RNAs that affectexpression by RNA interference, suppression of gene expression, oranother mechanism. Accordingly, in addition to shRNA, siRNA, miRNA, andantisense ssRNA, the category of regulatory RNAs includes ribozymes anddecoy RNAs, inter alia.

“Ribozyme” refers to an RNA molecule having an enzymatic activity thatcan repeatedly cleave other RNA molecules in a nucleotide basesequence-specific manner.

“RNA interference” (RNAi) denotes a RNA-guided mechanism as describedabove, involving degradation of complementary or partially complementarytarget RNA, for sequence- or gene-specific regulation of gene expression(protein synthesis).

“Sequence identity” connotes a degree of similarity or complementarity.There can be partial identity or complete identity. A partiallycomplementary sequence is one that at least partially inhibits anidentical sequence from hybridizing to a target polynucleotide; it isreferred to using the functional term “substantially identical.” Theinhibition of hybridization of the completely complementary sequence tothe target sequence can be examined using a hybridization assay(Southern or Northern blot, solution hybridization, and the like) underconditions of low stringency. A substantially identical sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely identical sequence or probe to the target sequence underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding can be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity); in the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

Another way of viewing sequence identity, in the context to two nucleicacid or polypeptide sequences, entails referencing residues in the twosequences that are the same when aligned for maximum correspondence overa specified region. As used herein, “percentage of sequence identity”means the value determined by comparing two optimally aligned sequencesover a comparison window, wherein the portion of the polynucleotidesequence in the comparison window can comprise additions or deletions(i.e., gaps) as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

“Short interfering RNA” (siRNA) refers to double-stranded RNA molecules,generally, from about 10 to about 30 nucleotides long that are capableof mediating RNA interference (RNAi). In general, siRNA molecules have acapacity to reduce expression of a protein by directly interacting witha transcript that encodes the protein.

A “therapeutically effective” amount of functional nucleic acid is adosage of the molecule in question, e.g., siRNA, miRNA or free shRNA,that invokes a pharmacological response when administered to a subject,in accordance with the present invention. In the context of the presentinvention, therefore, a therapeutically effective amount can be gaugedby reference to the prevention or amelioration of an adverse conditionor symptom associated with a disease or disorder, either in an animalmodel or in a human subject, when functional nucleic acid-packagedminicells are administered, as described in greater detail below. Anamount that proves “therapeutically effective amount” in a giveninstance, for a particular subject, may not be effective for 100% ofsubjects similarly treated for the disease or condition underconsideration, even though such dosage is deemed a “therapeuticallyeffective amount” by skilled practitioners. The appropriate dosage inthis regard also will vary as a function, for example, of the type,stage, and severity of the disease or condition to be affected. In anyevent, the present illustration of in vitro testing (Example 2) and invivo testing (Examples 4, 5 and 6) according to the present invention,as well as of methodology for quantifying a minicell-delivered amount ofan functional nucleic acid molecule (Example 3), when considered inlight of the entire description, empower a person knowledgeable inpre-clinical and clinical testing of drug candidates to determine,through routine experimentation, the therapeutically effective amount offunctional nucleic acid for a particular indication.

The terms “treatment,” “treating,” “treat,” and the like refer toobtaining a desired pharmacological and/or physiologic effect. Theeffect can be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or can be therapeutic interms of a partial or complete stabilization or cure for a diseaseand/or adverse effect attributable to the disease. “Treatment” coversany treatment of a disease in a mammal, particularly a human, andincludes: (a) preventing the disease or symptom from occurring in asubject which can be predisposed to the disease or symptom but has notyet been diagnosed as having it; (b) inhibiting the disease symptom,i.e., arresting its development; or (c) relieving the disease symptom,i.e., causing regression of the disease or symptom.

Minicells

Minicells of the invention are anucleate forms of E. coli or otherbacterial cells, engendered by a disturbance in the coordination, duringbinary fission, of cell division with DNA segregation. Prokaryoticchromosomal replication is linked to normal binary fission, whichinvolves mid-cell septum formation. In E. coli, for example, mutation ofmin genes, such as minCD, can remove the inhibition of septum formationat the cell poles during cell divisions resulting in production of anormal daughter cell and an anucleate minicell See de Boer et al., 1992;Raskin & de Boer, 1999; Hu & Lutkenhaus, 1999; Harry, 2001. Minicellsare distinct from other small vesicles that are generated and releasedspontaneously in certain situations and, in contrast to minicells, arenot due to specific genetic rearrangements or episomal gene expression.In a preferred embodiment, minicells possess intact cell walls (“intactminicells”).

In addition to min operon mutations, anucleate minicells also aregenerated following a range of other genetic rearrangements or mutationsthat affect septum formation, for example in the divIVB1 in B. subtilis.See Reeve and Cornett, 1975. Minicells also can be formed following aperturbation in the levels of gene expression of proteins involved incell division/chromosome segregation. For example, overexpression ofminE leads to polar division and production of minicells. Similarly,chromosome-less minicells can result from defects in chromosomesegregation for example the smc mutation in Bacillus subtilis (Brittonet al., 1998), spoOJ deletion in B. subtilis (Ireton et al., 1994), mukBmutation in E. coli (Hiraga et al., 1989), and parC mutation in E. coli(Stewart and D'Ari, 1992). Gene products can be supplied in trans. Whenover-expressed from a high-copy number plasmid, for example, CafA canenhance the rate of cell division and/or inhibit chromosome partitioningafter replication (Okada et al., 1994), resulting in formation ofchained cells and anucleate minicells (Wachi et al., 1989). Minicellscan be prepared from any bacterial cell of Gram-positive orGram-negative origin.

In one aspect, minicells can contain one or more plasmid-free functionalnucleic acid for which delivery is desired. Functional nucleic acid ofthe invention have the capacity to reduce expression of a protein bydirectly interacting with a transcript that encodes the protein.

Packaging Functional Nucleic Acid into Intact Minicells

Functional nucleic acid can be packaged directly into intact minicells.The process bypasses the previously required steps of, for example,cloning nucleic acids encoding functional nucleic acid into expressionplasmids, transforming minicell-producing parent bacteria with theplasmids and generating recombinant minicells. Instead, plasmid-freefunctional nucleic acid can be packaged directly into intact minicellsby co-incubating a plurality of intact minicells with functional nucleicacid in a buffer. In some embodiments, the co-incubation may involvegentle shaking, while in others the co-incubation is static. Aco-incubation period of about one hour has proven sufficient, butshorter periods, such as about half an hour, also may be effective. Inone embodiment, the buffer comprises buffered saline, for example a 1×phosphate buffer solution. The buffered saline can be in gelatin form.In another embodiment, the co-incubation is conducted at a temperatureof about 4° C. to about 37° C.; about 20° C. to about 30° C.; about 25°C.; or about 37° C. In other aspects, the co-incubation can compriseabout 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹² or 10¹³ minicells. Specificparameters of temperature, time, buffer, minicell concentration, etc.can be optimized for a particular combination of conditions.

The success of this approach is startling because, for over fourdecades, practitioners have developed a variety of chemical andelectrochemical processes (reviewed by Miller, 1994) to transformnucleic acids into bacterial cells. Practitioners have utilized suchharsh measures because conventional wisdom has held that nucleic acidssuch as siRNA, miRNA or plasmid-free shRNA are too large to passivelyenter into the minicell cytoplasm. For example, porins, which areβ-barrel proteins that typically function as diffusion pores, permitpassive transport across the bacterial outer membrane of molecules withmolecular weights of 600 daltons or less (Nikaido, 1994). Meanwhile,double stranded plasmid DNA encoding shRNA exceeds a million daltons,and double stranded siRNA or miRNA exceeds 15,000 daltons.

Moreover, once packaged, the functional nucleic acid remain inside theminicell and are protected from degradation. In this regard, prolongedincubation studies with siRNA-packaged minicells incubated in sterilesaline showed no leakage of siRNAs. In addition, co-incubatingsiRNA-packaged minicells with nucleases confirmed that the siRNAs hadpenetrated the outer membrane of the intact minicells and were protectedfrom degradation. Similarly, despite the fact that minicells might beexpected to carry residual nucleases from the parent bacterialcytoplasm, packaged siRNA are stable in the minicell cytoplasm. PackagedsiRNA also avoid the degradative machinery present withinphagolysosomes, such as acids, free oxygen radicals and acid hydrolases(Conner and Schmid, 2003), to effect target mRNA knockdown within themammalian cell.

In other embodiments, multiple functional nucleic acids directed todifferent mRNA targets can be packaged in the same minicell. Such anapproach can be used to combat drug resistance and apoptosis resistance.For example, cancer patients routinely exhibit resistance tochemotherapeutic drugs. Such resistance can be mediated byover-expression of genes such as multi-drug resistance (MDR) pumps andanti-apoptotic genes, among others. To combat this resistance, minicellscan be packaged with therapeutically significant concentrations offunctional nucleic acid to MDR-associated genes and administered to apatient before chemotherapy. Furthermore, packaging into the sameminicell multiple functional nucleic acid directed to different mRNAtargets can enhance therapeutic success since most molecular targets aresubject to mutations and have multiple alleles.

Thus, packaging plasmid-free functional nucleic acid directly intointact minicells, as described here, offers numerous advantages. Forexample, since the inventive approach does not require geneticallymodifying parent bacteria to accommodate expression of functionalnucleic acid, one parent bacteria can be used to produce minicellscomprising many types of nucleic acids, directed to a variety ofindications. Similarly, a minicell can be loaded with a variety ofdifferent RNAs, thereby to avoid or overcome resistance mechanisms.

Functional Nucleic Acids

As noted above, functional nucleic acid denotes a category inclusive ofnucleic acid molecules that affect expression by RNA interference,suppression of gene expression, or another mechanism. Such molecules areexemplified by single-, double-, or multi-stranded DNA or RNA. Examplesof functional nucleic acids include, but are not limited to regulatoryRNA, such as shRNA, siRNA, miRNA, and antisense ssRNA, therefore,ribozymes and decoy RNAs and antisense nucleic acids.

In a preferred embodiment of the invention, the intact minicells carrysiRNA molecules. Short interfering RNA molecules are useful forperforming RNAi, a post-transcriptional gene silencing mechanism. Asnoted, “siRNA” generally refers to double-stranded RNA molecules fromabout 10 to about 30 nucleotides long that are named for their abilityspecifically to interfere with protein expression. Preferably, siRNAmolecules are 12-28 nucleotides long, more preferably 15-25 nucleotideslong, still more preferably 19-23 nucleotides long and most preferably21-23 nucleotides long. Therefore, preferred siRNA molecules are 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28 or 29nucleotides in length.

The length of one strand designates the length of an siRNA molecule. Forinstance, an siRNA that is described as 21 ribonucleotides long (a21-mer) could comprise two opposite strands of RNA that anneal togetherfor 19 contiguous base pairings. The two remaining ribonucleotides oneach strand would form an “overhang.” When an siRNA contains two strandsof different lengths, the longer of the strands designates the length ofthe siRNA. For instance, a dsRNA containing one strand that is 21nucleotides long and a second strand that is 20 nucleotides long,constitutes a 21-mer.

siRNAs that comprise an overhang are desirable. The overhang can be atthe 5′ or the 3′ end of a strand. Preferably, it is at the 3′ end of theRNA strand. The length of an overhang can vary, but preferably is about1 to about 5 bases, and more preferably is about 2 nucleotides long.Preferably, the siRNA of the present invention will comprise a 3′overhang of about 2 to 4 bases. More preferably, the 3′ overhang is 2ribonucleotides long. Even more preferably, the 2 ribonucleotidescomprising the 3′ overhang are uridine (U).

shRNAs comprise a single strand of RNA that forms a stem-loop structure,where the stem consists of the complementary sense and antisense strandsthat comprise a double-stranded siRNA, and the loop is a linker ofvarying size. The stem structure of shRNAs generally is from about 10 toabout 30 nucleotides long. Preferably, the stem of shRNA molecules are12-28 nucleotides long, more preferably 15-25 nucleotides long, stillmore preferably 19-23 nucleotides long and most preferably 21-23nucleotides long. Therefore, preferred shRNA molecules comprise stemsthat are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 2728 or 29 nucleotides in length.

siRNAs of the invention are designed to interact with a targetribonucleotide sequence, meaning they complement a target sequencesufficiently to hybridize to the target sequence. In one embodiment, theinvention provides an siRNA molecule comprising a ribonucleotidesequence at least 70%, 75%, 80%, 85% or 90% identical to a targetribonucleotide sequence or the complement of a target ribonucleotidesequence. Preferably, the siRNA molecule is at least 90%, 95%, 96%, 97%,98%, 99% or 100% identical to the target ribonucleotide sequence or thecomplement of the target ribonucleotide sequence. Most preferably, ansiRNA will be 100% identical to the target nucleotide sequence or thecomplement of the ribonucleotide sequence. However, siRNA molecules withinsertions, deletions or single point mutations relative to a targetalso can be effective.

Accordingly, in one aspect of the invention, intact minicells can carryone or more siRNA sequences aimed at silencing drug resistance orapoptosis resistance genes. Using minicells that encode multiple siRNAs,it is possible to treat cells that express multiple drug resistancemechanisms.

Tools to assist siRNA design, and regulatory RNA in general, are readilyavailable to the public. For example, a computer-based siRNA design toolis available on the internet at www.dharmacon.com.

Targets of Functional Nucleic Acid

Functional nucleic acid of the invention target the gene or transcriptof a protein that promotes drug resistance, inhibits apoptosis, promotesa neoplastic phenotype, inhibits pathogen proliferation or inhibitsviral replication or proliferation. Successful application of functionalnucleic acid strategies in these contexts have been achieved in the art,but without the benefits of minicell vectors. See, e.g., Sioud (2004),Caplen (2003), Wu et al. (2003), Yague et al. (2004).

Proteins that contribute to drug resistance or promotion of neoplasticphenotype constitute preferred targets of functional nucleic acid. Theproteins can contribute to acquired drug resistance or intrinsic drugresistance. When diseased cells, such as tumor cells, initially respondto drugs, but become refractory on subsequent treatment cycles, theresistant phenotype is acquired. Useful targets involved in acquired orintrinsic drug resistance include but not limited to ATP bindingcassette transporters such as P-glycoprotein (P-gp, P-170, PGY1, MDR1,ABCB1, MDR-associated protein, Multidrug resistance protein 1, MDR-2 andMDR-3, MRP2 (multi-drug resistance associated protein), BCR-ABL(breakpoint cluster region—Abelson protooncogene), STI-571resistance-associated protein, lung resistance-related protein,cyclooxygenase-2, nuclear factor kappa, XRCC1 (X-ray cross-complementinggroup 1), ERCC1 (Excision cross-complementing gene), GSTP1 (GlutathioneS-transferase), mutant β-tubulin, Abcb1a (ABCB4), Abcc1, Abcc2, Abcc3(MLP-2), Abcc5, Abcc6, Abcd2, Abcg2, Bax, Bcl2, Bcl21 (bcl-x), Mvp, Rb1,Top1, Top2a, Top2b, Trp53 (p53). Other genes involved in drug resistancealso include (a) genes involved in drug metabolism for example Arnt,Blmh, C130052I12Rik (CRR9p), Comt, Crabp1. Cyp1a1, Cyp1a2, Cyp2b 9,Cyp2b20, Cyp2c29, Cyp2c40, Cyp2c70, Cyp2d22, Cyp2e1, Dhfr, Ephx1, Ephx2,Gstm1 (MGST1), Gstp1, Nat2, Nqo1, Sod1, Ste, Tpmt, Tyms, Ugcg, (b) genesinvolved in DNA repair for example Apc, Atm, Brca1, Brca2, Ercc3 (XPB),Mgmt, Mlh1, Xpa, Xpc, (c) genes involved in cell cycle for example Ccnd1(cyclin D1), Ccne1 (cyclin E1), Cdk1, Cdk2, Cdk4, Cdkn1a (p21Waf1),Cdkn1b (p27Kip1), Cdkn2a (p16Ink4a), Cdkn2d (p19), KSP, (d) genesinvolved in growth factor receptors for example Egfr, Erbb2 (Neu, HER2),Erbb3, Erbb4, Fgf2 (bFGF), Met, (e) genes involved in hormone receptorsfor example Ar, Esr1, Esr2, Igf2r, Ppara, Ppard, Pparg, Ppargc1, Rara,Rarb, Rxra, Rxrb, Rxrg, Srd5a2, and (f) genes involved in transcriptionfactors for example Ahr, Apls1, Apls2, Elk1, Fos (c-fos), Gabpa, Hif1a,Mafb, Myc (c-myc), Nfkb1, Nfkb2, Nfkbib, Nfkbie, Relb (1-rel),Tnfrsfl1A.

Useful targets also include proteins that contribute to apoptosisresistance. These include Bcl-2 (B cell leukemia/lymphoma), Bcl-X_(L),A1/Bfl 1, focal adhesion kinase and p53 mutant protein.

Useful targets further include oncogenic and mutant tumor suppressorproteins. Examples include β-Catenin, PKC-α (protein kinase C), C-RAF,K-Ras (V12), h-Ras, DP97 Dead box RNA helicase, DNMT1 (DNAmethyltransferase 1), FLIP (Flice-like inhibitory protein), C-Sfc,53BPI, Polycomb group protein EZH2 (Enhancer of zeste homologue), ErbB1,HPV-16 E5 and E7 (human papillomavirus early 5 and early 7), Fortilin &MCI1P (Myeloid cell leukemia 1 protein), DlP13α (DDC interacting protein13a), MBD2 (Methyl CpG binding domain), p21, KLF4 (Kruppel-like factor4), tpt/TCTP (Translational controlled tumor protein), SPK1 & SPK2(Sphingosine kinase), P300, PLK1 (Polo-like kinase-1), Trp53, Ras,ErbB1, VEGF (Vascular endothelial growth factor), and BAG-1(BCL2-associated athanogene 1).

A large number of molecular targets have been identified for thetreatment of cancer, and RNAi discovery platforms are rapidlyidentifying a range of different new targets. Examples of such moleculartargets useful in this invention include, tyrosine kinase (variant), Akt(protein kinase B, PKB), Akt1, AlphaLbeta2 integrin, Aminopeptidase,Androgen receptor, Aurora A, AuroraB, Basic fibroblast growth factor(bFGF) receptor (bFGFr), BRaf, Carcinoembryonic antigen (CEA), CD142,CD37, CD44, CD5, CD74, CD77, Chk1, CHK2, CHras, CSF1r, CXCR4, Cyclin D1(CCND1), Cyclin-dependent kinase 1 (CDK1), Cyclin dependent kinase 2(CDK2), Cyclin-dependent kinase inhibitor 1B (CDKN1B, p27, KIP1), CYP26,Fibroblast growth factor receptor 3 (FGFr3), Fibroblast growth factorreceptor 4 (FGFr4), G250, Hedgehog (Hh) signaling pathway, Hepatocytegrowth factor/scatter factor (HGF/SF or SF/HGF), HEr4 (ErbB4), HIF,Histone deacetylase 9 (HDAC9), Homeobox gene (HOXB7), Hyaluronan (HA),Insulin like growth factor (IGF), Insulin like growth factor 1 receptor(IGF1r, IGF1r, IGF1r, IGF1r), Insulin like growth factor binding protein2 (IGFBP2), Insulin like growth factor binding protein 5 (IGFBP5),Integrin like kinase (ILK) Interleukin (IL6) receptor, Interleukin 1(IL1) receptor type II, Interleukin 10 (IL10), Interleukin 4 (IL4)receptor, Interleukin 6 (IL6), Interleukin15 (IL15), Interleukin3receptor alpha (IL3r alpha) chain, JAK, JAK3, JNK1, JNK2, KinesinSpindle Protein (KSP), Laminin 5, Lewis (b), Lymphotoxin (LT) betareceptor (LTBr), Lysophosphatidic acid (LPA) receptors (LPAr),Lysophosphatidic acid acyltransferase, Macrophage migration inhibitoryfactor (MIF), MAGE3, Microtubules, MUC2, Notch 1 (TAN1), P38 mitogenactivated protein kinase (p38 MAPK), P53 upregulated mediator ofapoptosis (PUMA), PDGF tyrosine kinase (TK) signaling pathway,Phosphatase and tensin homolog (PTEN), Phosphatidylinositol 3′kinase(PI3K), Plasminogen activator, urokinase (PLAU) receptor (PLAUr),Pololike kinase 1 (Plk1), Poly (ADP ribose), polymerase (PARP),Proliferating cell nuclear antigen (PCNA), Prostate stem cell antigen(PSCA), Prostate specific antigen (PSA) 773, Protein tyrosinephosphatase (PTP), Rad51 protein, RAF1, Retinoic acid receptor (RAr)alpha, Retinoic acid receptor (RAr) gamma, Retinoid X receptor (RXr)beta, Serine (or cysteine) proteinase inhibitor, Telomerase reversetranscriptase (TERT, hTERT), Telomeres, Thomsen Friedenreich (TF)antigen, Thrombospondinl (TSP1), Transferrin, Tumor necrosis factoralpha (TNFa, TNFA), Tumor necrosis factor receptor (TNFr, TNFr), Tumorassociated carbonic anhydrase (CA) IX (CA9), Type I interferon,Ubiquitin ligase, Vascular cell adhesion molecule 1 (VCAM1, CD106),Vascular endothelial growth factor (VEGF, VEGFA), Vascular endothelialgrowth factor D (VEGFD), Vitronectin (VTN), Wilms' tumor 1 (WT1) etc.

With regard to HIV infection, targets include HIV-Tat, HIV-Rev, HIV-Vif,HIV-Nef, HIV-Gag, HIV-Env, LTR, CD4, CXCR4 (chemokine receptor) and CCR5(chemokine receptor).

Because of tumor cell heterogeneity, a number of different drugresistance or apoptosis resistance pathways can be operational in targetcells. Therefore, the functional nucleic acid used in methods of theinvention can require change over time. For instance, if biopsy samplesreveal new mutations that result in acquired drug resistance, specificfunctional nucleic acid can be designed and packaged into intactminicells that are administered to the mammalian host to address theacquired drug resistance.

Delivery of Functional Nucleic Acid via Intact Minicells

In a second aspect, the invention provides a method of deliveringfunctional nucleic acid, comprising (a) providing a plurality of intactminicells in a pharmaceutically acceptable carrier, each minicell of theplurality encompassing plasmid-free functional nucleic acid, and (b)bringing minicells of the plurality into contact with mammalian cellssuch that the mammalian cells engulf minicells of the plurality, wherebythe functional nucleic acid is released into the cytoplasm of the targetcells. Minicells are brought into contact with the target mammalian cellvia bispecific ligands as described in published PCT application WO05/056749. Contact between the minicell and the target mammalian cellcan be in vitro or in vivo.

Method of Overcoming Drug Resistance and Treating Disease

In another aspect, the invention provides a method of overcoming drugresistance and treating a disease, such as cancer or AIDS, in a subject.The method comprises (a) packaging one or more functional nucleic acidthat target genes or transcripts of proteins that promote drugresistance into intact purified minicells, (b) bringing the functionalnucleic acid containing minicells into contact with a target mammaliancell, such that the mammalian cell engulfs the minicell, as described inthe above-cited '749 PCT application, which is hereby incorporated byreference, and (c) delivering a drug to the target mammalian cell, asdescribed in published PCT application WO 05/079854. Preferably, step(c) is performed after steps (a) and (b), to allow the functionalnucleic acid to diminish resistance to the drug prior to the drug'sadministration. Delivery of the drug and introduction of the functionalnucleic acid can occur consecutively, in any order, or simultaneously.

According to the invention, drugs can be delivered by any conventionalmeans. For example, drugs can be delivered orally, parenterally(including subcutaneously, intravenously, intramuscularly,intraperitoneally, and by infusion), topically, transdermally or byinhalation. The appropriate mode of delivery and dosage of each drug iseasily ascertainable by those skilled in the medical arts.

Drug Delivery Via Minicells

Although drug delivery can occur via conventional means, delivery viaminicells is preferred, as described in published PCT application WO05/079854, which is hereby incorporated by reference. In this regard,the inventors have discovered that the same mammalian cells can besuccessfully re-transfected by targeted intact minicells that arepackaged with different payloads. For example, functional nucleicacid-packaged minicells can transfect a mammalian cell, after whichdrug-packaged minicells can deliver drug to the same mammalian cell toobtain a complementary or synergistic anti-tumor effect.

The drug can be packaged in a separate minicell from the functionalnucleic acid. Alternatively, the drug can be packaged in the sameminicell as the functional nucleic acid. Certain drugs can interact withnucleic acids and preclude co-packaging of drug and nucleic acid in thesame minicell. For example, Doxorubicin is known to interact with DNA.

Preferably, minicells of the invention contain a sufficient quantity ofdrug to exert the drug's physiological or pharmacological effect on atarget cell. Also preferably, drugs contained within the minicells areheterologous, or foreign, to the minicells, meaning that the minicells'parent bacterial cells do not normally produce the drug.

Both hydrophilic and hydrophobic drugs can be packaged in minicells bycreating a concentration gradient of the drug between an extracellularmedium containing minicells and the minicell cytoplasm. When theextracellular medium contains a higher drug concentration than theminicell cytoplasm, the drug naturally moves down this concentrationgradient, into the minicell cytoplasm. When the concentration gradientis reversed, however, the drug does not move out of the minicells. Theprocedure and mechanisms for drug loading into minicells is as describedin published PCT application WO 05/079854.

To load minicells with drugs that normally are not water soluble, thedrugs initially can be dissolved in an appropriate solvent. For example,Paclitaxel can be dissolved in a 1:1 blend of ethanol and cremophore EL(polyethoxylated castor oil), followed by a dilution in PBS to achieve asolution of Paclitaxel that is partly diluted in aqueous media andcarries minimal amounts of the organic solvent to ensure that the drugremains in solution. Minicells can be incubated in this final medium fordrug loading. Thus, the inventors discovered that even hydrophobic drugscan diffuse into the cytoplasm of minicells to achieve a high andtherapeutically significant cytoplasmic drug load. This is unexpectedbecause the minicell membrane is composed of a hydrophobic phospholipidbilayer, which would be expected to prevent diffusion of hydrophobicmolecules into the cytoplasm.

Another method of loading minicells with a drug involves culturing arecombinant parent bacterial cell under conditions such that the parentbacterial cell transcribes and translates a nucleic acid encoding thedrug, and the drug is released into the cytoplasm of the parentbacterial cell. For example, a gene cluster encoding the cellularbiosynthetic pathway for a desired drug can be cloned and transferredinto a parent bacterial strain that is capable of producing minicells.Genetic transcription and translation of the gene cluster results inbiosynthesis of the drug within the cytoplasm of the parent bacterialcells, filling the bacterial cytoplasm with the drug. When the parentbacterial cell divides and forms progeny minicells, the minicells alsocontain the drug in their cytoplasm. The pre-packaged minicells can bepurified by any suitable minicell-purification process, including themethodology described above.

Similarly, another method of loading minicells with a drug involvesculturing a recombinant minicell that contains an expression plasmidencoding the drug under conditions such that the gene encoding the drugis transcribed and translated within the minicell.

Drugs

Drugs useful in the invention can be any physiologically orpharmacologically active substance that produces a desired local orsystemic effect in animals, particularly mammals and humans. Drugs canbe inorganic or organic compounds, without limitation, includingpeptides, proteins, nucleic acids, and small molecules, any of which canbe characterized or uncharacterized. They can be in various forms, suchas unchanged molecules, molecular complexes, pharmacologicallyacceptable salts, such as hydrochloride, hydrobromide, sulfate, laurate,palmitate, phosphate, nitrite, nitrate, borate, acetate, maleate,tartrate, oleate, salicylate, and the like. For acidic drugs, salts ofmetals, amines or organic cations, for example, quaternary ammonium, canbe used. Derivatives of drugs, such as bases, esters and amides also canbe used. A drug that is water insoluble can be used in a form that is awater soluble derivative thereof, or as a base derivative thereof, whichin either instance, or by its delivery, is converted by enzymes,hydrolyzed by the body pH, or by other metabolic processes to theoriginal therapeutically active form.

Useful drugs include chemotherapeutic agents, immunosuppressive agents,cytokines, cytotoxic agents, nucleolytic compounds, radioactiveisotopes, receptors, and pro-drug activating enzymes, which can benaturally occurring or produced by recombinant methods.

Drugs that are affected by classical multidrug resistance haveparticular utility in the invention, such as vinca alkaloids (e.g.,vinblastine and vincristine), the anthracyclines (e.g., doxorubicin anddaunorubicin), RNA transcription inhibitors (e.g., actinomycin-D) andmicrotubule stabilizing drugs (e.g., paclitaxel).

In general, cancer chemotherapy agents are preferred drugs. Usefulcancer chemotherapy drugs include nitrogen mustards, nitrosorueas,ethyleneimine, alkane sulfonates, tetrazine, platinum compounds,pyrimidine analogs, purine analogs, antimetabolites, folate analogs,anthracyclines, taxanes, vinca alkaloids, topoisomerase inhibitors andhormonal agents. Exemplary chemotherapy drugs are Actinomycin-D,Alkeran, Ara-C, Anastrozole, Asparaginase, BiCNU, Bicalutamide,Bleomycin, Busulfan, Capecitabine, Carboplatin, Carboplatinum,Carmustine, CCNU, Chlorambucil, Cisplatin, Cladribine, CPT-11,Cyclophosphamide, Cytarabine, Cytosine arabinoside, Cytoxan,Dacarbazine, Dactinomycin, Daunorubicin, Dexrazoxane, Docetaxel,Doxorubicin, DTIC, Epirubicin, Ethyleneimine, Etoposide, Floxuridine,Fludarabine, Fluorouracil, Flutamide, Fotemustine, Gemcitabine,Herceptin, Hexamethylamine, Hydroxyurea, Idarubicin, Ifosfamide,Irinotecan, Lomustine, Mechlorethamine, Melphalan, Mercaptopurine,Methotrexate, Mitomycin, Mitotane, Mitoxantrone, Oxaliplatin,Paclitaxel, Pamidronate, Pentostatin, Plicamycin, Procarbazine,Rituximab, Steroids, Streptozocin, STI-571, Streptozocin, Tamoxifen,Temozolomide, Teniposide, Tetrazine, Thioguanine, Thiotepa, Tomudex,Topotecan, Treosulphan, Trimetrexate, Vinblastine, Vincristine,Vindesine, Vinorelbine, VP-16, and Xeloda.

Useful cancer chemotherapy drugs also include alkylating agents such asThiotepa and cyclosphosphamide; alkyl sulfonates such as Busulfan,Improsulfan and Piposulfan; aziridines such as Benzodopa, Carboquone,Meturedopa, and Uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as Chlorambucil, Chlornaphazine, Cholophosphamide,Estramustine, Ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, Melphalan, Novembiehin, Phenesterine, Prednimustine,Trofosfamide, uracil mustard; nitroureas such as Cannustine,Chlorozotocin, Fotemustine, Lomustine, Nimustine, and Ranimustine;antibiotics such as Aclacinomysins, Actinomycin, Authramycin, Azaserine,Bleomycins, Cactinomycin, Calicheamicin, Carabicin, Caminomycin,Carzinophilin, Chromoinycins, Dactinomycin, Daunorubicin, Detorubicin,6-diazo-5-oxo-L-norleucine, Doxorubicin, Epirubicin, Esorubicin,Idambicin, Marcellomycin, Mitomycins, mycophenolic acid, Nogalamycin,Olivomycins, Peplomycin, Potfiromycin, Puromycin, Quelamycin,Rodorubicin, Streptonigrin, Streptozocin, Tubercidin, Ubenimex,Zinostatin, and Zorubicin; anti-metabolites such as Methotrexate and5-fluorouracil (5-FU); folic acid analogues such as Denopterin,Methotrexate, Pteropterin, and Trimetrexate; purine analogs such asFludarabine, 6-mercaptopurine, Thiamiprine, and Thioguanine; pyrimidineanalogs such as Ancitabine, Azacitidine, 6-azauridine, Carmofur,Cytarabine, Dideoxyuridine, Doxifluridine, Enocitabine, Floxuridine, and5-FU; androgens such as Calusterone, Dromostanolone Propionate,Epitiostanol, Rnepitiostane, and Testolactone; anti-adrenals such asaminoglutethimide, Mitotane, and Trilostane; folic acid replenisher suchas frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine;Demecolcine; Diaziquone; Elformithine; elliptinium acetate; Etoglucid;gallium nitrate; hydroxyurea; Lentinan; Lonidamine; Mitoguazone;Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenarnet;Pirarubicin; podophyllinic acid; 2-ethylhydrazide; Procarbazine; PSK®;Razoxane; Sizofrran; Spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; Urethan; Vindesine; Dacarbazine;Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine;Arabinoside (“Ara-C”); cyclophosphamide; thiotEPa; taxoids, e.g.,Paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) andDoxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France);Chlorambucil; Gemcitabine; 6-thioguanine; Mercaptopurine; Methotrexate;platinum analogs such as Cisplatin And Carboplatin; Vinblastine;platinum; etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone;Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin;Aminopterin; Xeloda; Ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; Esperamicins;Capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included are anti-hormonal agentsthat act to regulate or inhibit hormone action on tumors such asanti-estrogens including for example Tamoxifen, Raloxifene, aromataseinhibiting 4(5)-imidazoles, 4 Hydroxytamoxifen, Trioxifene, Keoxifene,Onapristone, And Toremifene (Fareston); and anti-androgens such asFlutamide, Nilutamide, Bicalutamide, Leuprolide, and Goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Useful drugs also include cytokines. Examples of such cytokines arelymphokines, monokines, and traditional polypeptide hormones. Includedamong the cytokines are growth hormones such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (GCSF); interleukins (ILs) such as IL-1, IL-1a, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumornecrosis factor such as TNF-α or TNF-β; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the tern cytokineincludes proteins from natural sources or from recombinant cell cultureand biologically active equivalents of the native sequence cytokines.

The drugs can be prodrugs, subsequently activated, e.g., by aprodrug-activating enzyme that converts a prodrug, such as a peptidylchemotherapeutic agent, to an active anti-cancer drug. For instance, seeWO 88/07378, WO 81/01145, and U.S. Pat. No. 4,975,278. In general, theenzyme component includes any enzyme capable of acting on a prodrug insuch a way so as to covert it into its more active, cytotoxic form.

Directing Minicells to Specific Mammalian Cells

In one aspect of the invention, a minicell is directed to a targetmammalian cell via a bispecific ligand as described in published PCTapplications WO 05/056749 and WO 05/079854. The bispecific ligand,having specificity for both minicell and mammalian cell components,causes the minicell to bind to the mammalian cell, such that theminicell is engulfed by the mammalian cell, whereby functional nucleicacid is released into the cytoplasm of the mammalian cell. This targeteddelivery method can be performed in vivo or in vitro, or both in vivoand in vitro.

Contact between bispecific ligand, minicell and mammalian cell can occurin a number of different ways. For in vivo delivery, it is preferable toadminister a minicell that already has the bispecific ligand attached toit. Thus, minicell, bispecific ligand and target cell all are broughtinto contact when the bispecific ligand-targeted minicell reaches thetarget cell in vivo. Alternatively, bispecific ligand and minicell canbe separately administered in vivo.

Contact between the bispecific ligands, minicells and mammalian cellsalso can occur during one or more incubations in vitro. In oneembodiment, the three elements are incubated together all at once.Alternatively, step-wise incubations can be performed. In one example ofa step-wise approach, minicells and bi-specific ligands are firstincubated together to form bispecific ligand-targeted minicells, whichare then incubated with target cells. In another example, bispecificligands are first incubated with target cells, followed by an incubationwith minicells. A combination of one or more in vitro incubations and invivo administrations also can bring bispecific ligands, minicells andmammalian target cells into contact.

The inventors found that the targeted delivery approach is broadlyapplicable to a range of mammalian cells, including cells that normallyare refractory to specific adhesion and endocytosis of minicells. Forexample, bispecific antibody ligands with anti-O-polysaccharidespecificity on one arm and anti-HER2 receptor or anti-EGF receptorspecificity on the other arm efficiently bind minicells to therespective receptors on a range of target non-phagocytic cells. Thesecells include lung, ovarian, brain, breast, prostate and skin cancercells. Moreover, the efficient binding precedes rapid endocytosis of theminicells by each of the non-phagocytic cells.

Target cells of the invention include any cell into which a functionalnucleic acid is to be introduced. Desirable target cells arecharacterized by expression of a cell surface receptor that, uponbinding of a ligand, facilitates endocytosis. Preferred target cells arenon-phagocytic, meaning that the cells are not professional phagocytes,such as macrophages, dendritic cells and Natural Killer (NK) cells.Preferred target cells also are mammalian.

Ligands useful in the targeted delivery methods of this inventioninclude any agent that binds to a surface component on a target cell andto a surface component on a minicell. Preferably, the surface componenton a target cell is a receptor, especially a receptor capable ofmediating endocytosis. The ligands can comprise a polypeptide and/orcarbohydrate component. Antibodies are preferred ligands. For example, abispecific antibody that carries dual specificities for a surfacecomponent on bacterially derived intact minicells and for a surfacecomponent on target mammalian cells, can be used efficiently to targetthe minicells to the target mammalian cells in vitro and in vivo. Usefulligands also include receptors, enzymes, binding peptides,fusion/chimeric proteins and small molecules.

The selection of a particular ligand is made on two primary criteria:(i) specific binding to one or more domains on the surface of intactminicells and (ii) specific binding to one or more domains on thesurface of the target cells. Thus, ligands preferably have a first armthat carries specificity for a bacterially derived intact minicellsurface structure and a second arm that carries specificity for amammalian cell surface structure. Each of the first and second arms canbe multivalent. Preferably, each arm is monospecific, even ifmultivalent.

For binding to bacterially derived minicells, it is desirable for onearm of the ligand to be specific for the O-polysaccharide component of alipopolysaccharide found on the parent bacterial cell. Other minicellsurface structures that can be exploited for ligand binding include cellsurface-exposed polypeptides and carbohydrates on outer membranes, suchas outer-membrane proteins, pilli, fimbrae and flagella cell surfaceexposed peptide segments.

For binding to target cells, one arm of the ligand is specific for asurface component of a mammalian cell. Such components include cellsurface proteins, peptides and carbohydrates, whether characterized oruncharacterized. Cell surface receptors, especially those capable ofactivating receptor-mediated endocytosis, are desirable cell surfacecomponents for targeting. Such receptors, if over-expressed on thetarget cell surface, confer additional selectivity for targeting thecells to be treated, thereby reducing the possibility for delivery tonon-target cells.

By way of example, one can target tumor cells, metastatic cells,vasculature cells, such as endothelial cells and smooth muscle cells,lung cells, kidney cells, blood cells, bone marrow cells, brain cells,liver cells, and so forth, or precursors of any selected cell byselecting a ligand that specifically binds a cell surface receptor motifon the desired cells. Examples of cell surface receptors includecarcinoembryonic antigen (CEA), which is overexpressed in most colon,rectum, breast, lung, pancreas and gastrointestinal tract carcinomas(Marshall, 2003); heregulin receptors (HER-2, neu or c-erbB-2), which isfrequently overexpressed in breast, ovarian, colon, lung, prostate andcervical cancers (Hung et al., 2000); epidermal growth factor receptor(EGFR), which is highly expressed in a range of solid tumors includingthose of the breast, head and neck, non-small cell lung and prostate(Salomon et al., 1995); asialoglycoprotein receptor (Stockert, 1995);transferrin receptor (Singh, 1999); serpin enzyme complex receptor,which is expressed on hepatocytes (Ziady et al., 1997); fibroblastgrowth factor receptor (FGFR), which is overexpressed on pancreaticductal adenocarcinoma cells (Kleeff et al., 2002); vascular endothelialgrowth factor receptor (VEGFR), for anti-angiogenesis gene therapy(Becker et al., 2002; Hoshida et al., 2002); folate receptor, which isselectively overexpressed in 90% of nonmucinous ovarian carcinomas(Gosselin and Lee, 2002); cell surface glycocalyx (Batra et al., 1994);carbohydrate receptors (Thurnher et al., 1994); and polymericimmunoglobulin receptor, which is useful for gene delivery torespiratory epithelial cells and attractive for treatment of lungdiseases such as Cystic Fibrosis (Kaetzel et al., 1997).

Preferred ligands comprise antibodies and/or antibody derivatives. Asused herein, the term “antibody” encompasses an immunoglobulin moleculeobtained by in vitro or in vivo generation of an immunogenic response.The term “antibody” includes polyclonal, monospecific and monoclonalantibodies, as well as antibody derivatives, such as single-chainantibody fragments (scFv). Antibodies and antibody derivatives useful inthe present invention also can be obtained by recombinant DNAtechniques.

Wild-type antibodies have four polypeptide chains, two identical heavychains and two identical light chains. Both types of polypeptide chainshave constant regions, which do not vary or vary minimally amongantibodies of the same class, and variable regions. Variable regions areunique to a particular antibody and comprise an antigen binding domainthat recognizes a specific epitope. The regions of the antigen bindingdomain that are most directly involved in antibody binding are“complementarity-determining regions” (CDRs).

The term “antibody” also encompasses derivatives of antibodies, such asantibody fragments that retain the ability to specifically bind toantigens. Such antibody fragments include Fab fragments (a fragment thatcontains the antigen-binding domain and comprises a light chain and partof a heavy chain bridged by a disulfide bond), Fab′ (an antibodyfragment containing a single antigen-binding domain comprising a Fab andan additional portion of the heavy chain through the hinge region,F(ab′)2 (two Fab′ molecules joined by interchain disulfide bonds in thehinge regions of the heavy chains), a bispecific Fab (a Fab moleculehaving two antigen binding domains, each of which can be directed to adifferent epitope), and an scFv (the variable, antigen-bindingdeterminative region of a single light and heavy chain of an antibodylinked together by a chain of amino acids.)

When antibodies, including antibody fragments, constitute part or all ofthe ligands, they preferably are of human origin or are modified to besuitable for use in humans. So-called “humanized antibodies” are wellknown in the art. See, e.g., Osbourn et al., 2003. They have beenmodified by genetic manipulation and/or in vitro treatment to reducetheir antigenicity in a human. Methods for humanizing antibodies aredescribed, e.g., in U.S. Pat. No. 6,639,055, U.S. Pat. No. 5,585,089,and U.S. Pat. No. 5,530,101. In the simplest case, humanized antibodiesare formed by grafting the antigen-binding loops, known ascomplementarity-determining regions (CDRs), from a mouse mAb into ahuman IgG. See Jones et al., 1986; Riechmann et al., 1988; Verhoeyen etal., 1988. The generation of high-affinity humanized antibodies,however, generally requires the transfer of one or more additionalresidues from the so-called framework regions (FRs) of the mouse parentmAb. Several variants of the humanization technology also have beendeveloped. See Vaughan et al., 1998.

Human antibodies, rather than “humanized antibodies,” also can beemployed in the invention. They have high affinity for their respectiveantigens and are routinely obtained from very large, single-chainvariable fragments (scFvs) or Fab phage display libraries. See Griffithset al., 1994; Vaughan et al., 1996; Sheets et al., 1998; de Haard etal., 1999; and Knappik et al., 2000.

Useful ligands also include bispecific single chain antibodies, whichtypically are recombinant polypeptides consisting of a variable lightchain portion covalently attached through a linker molecule to acorresponding variable heavy chain portion. See U.S. Pat. No. 5,455,030,U.S. Pat. No. 5,260,203, and U.S. Pat. No. 4,496,778. Bispecificantibodies also can be made by other methods. For example, chemicalheteroconjugates can be created by chemically linking intact antibodiesor antibody fragments of different specificities. See Karpovsky et al.,1984. However, such heteroconjugates are difficult to make in areproducible manner and are at least twice as large as normal monoclonalantibodies. Bispecific antibodies also can be created by disulfideexchange, which involves enzymatic cleavage and reassociation of theantibody fragments. See Glennie et al., 1987.

Because Fab and scFv fragments are monovalent they often have lowaffinity for target structures. Therefore, preferred ligands made fromthese components are engineered into dimeric, trimeric or tetramericconjugates to increase functional affinity. See Tomlinson and Holliger,2000; Carter, 2001; Hudson and Souriau, 2001; and Todorovska et al.,2001. Such conjugate structures can be created by chemical and/orgenetic cross-links.

Bispecific ligands of the invention preferably are monospecific at eachend, i.e., specific for a single component on minicells at one end andspecific for a single component on target cells at the other end. Theligands can be multivalent at one or both ends, for example, in the formof so-called diabodies, triabodies and tetrabodies. See Hudson andSouriau, 2003. A diabody is a bivalent dimer formed by a non-covalentassociation of two scFvs, which yields two Fv binding sites. Likewise, atriabody results from the formation of a trivalent trimer of threescFvs, yielding three binding sites, and a tetrabody results from theformation of a tetravalent tetramer of four scFvs, yielding four bindingsites.

Several humanized, human, and mouse monoclonal antibodies and fragmentsthereof that have specificity for receptors on mammalian cells have beenapproved for human therapeutic use, and the list is growing rapidly. SeeHudson and Souriau, 2003. An example of such an antibody that can beused to form one arm of a bispecific ligand has specificity for HER2:Herceptin™; Trastuzumab.

Antibody variable regions also can be fused to a broad range of proteindomains. Fusion to human immunoglobulin domains such as IgG1 CH3 bothadds mass and promotes dimerization. See Hu et al., 1996. Fusion tohuman Ig hinge-Fc regions can add effector functions. Also, fusion toheterologous protein domains from multimeric proteins promotesmultimerization. For example, fusion of a short scFv to shortamphipathic helices has been used to produce miniantibodies. See Packand Pluckthun, 1992. Domains from proteins that form heterodimers, suchas fos/jun, can be used to produce bispecific molecules (Kostelny etal., 1992) and, alternately, homodimerization domains can be engineeredto form heterodimers by engineering strategies such as “knobs intoholes” (Ridgway et al., 1996). Finally, fusion protein partners can beselected that provide both multimerization as well as an additionalfunction, e.g. streptavidin. See Dubel et al., 1995.

Delivery to Phagocytosis- or Endocytosis-Competent Cells

The invention further provides for delivery by means of bringingbacterially derived minicells into contact with mammalian cells that arephagocytosis- or endocytosis-competent. Such mammalian cells, which arecapable of engulfing parent bacterial cells in the manner ofintracellular bacterial pathogens, likewise engulf the minicells, whichrelease their payload into the cytoplasm of the mammalian cells. Thisdelivery approach can be effected without the use of targeting ligands.

A variety of mechanisms can be involved in the engulfing of minicells bya given type of cell, and the present invention is not dependent on anyparticular mechanism in this regard. For example, phagocytosis is awell-documented process in which macrophages and other phagocyte cells,such as neutrophils, ingest particles by extending pseudopodia over theparticle surface until the particle is totally enveloped. Althoughdescribed as “non-specific” phagocytosis, the involvement of specificreceptors in the process has been demonstrated. See Wright et al.,(1986); Speert et al., (1988).

Thus, one form of phagocytosis involves interaction between surfaceligands and ligand-receptors located at the membranes of thepseudopodia. This attachment step, mediated by the specific receptors,is thought to be dependent on bacterial surface adhesins. With respectto less virulent bacteria, such as non-enterotoxigenic E. coli,phagocytosis also can occur in the absence of surface ligands forphagocyte receptors. See Pikaar et al. (1995), for instance. Thus, thepresent invention encompasses but is not limited to the use of minicellsthat either possess or lack surface adhesins, in keeping with the natureof their parent bacterial cells, and are engulfed by phagocytes (i.e.,“phagocytosis-competent” host cells), of which neutrophils andmacrophages are the primary types in mammals.

Another engulfing process is endocytosis, by which intracellularpathogens exemplified by species of Salmonella, Escherichia, Shigella,Helicobacter, Pseudomonas and Lactobacilli gain entry to mammalianepithelial cells and replicate there. Two basic mechanisms in thisregard are Clathrin-dependent receptor-mediated endocytosis, also knownas “coated pit endocytosis” (Riezman, 1993), and Clathrin-independentendocytosis (Sandvig & Deurs, 1994). Either or both can be involved whenan engulfing-competent cell that acts by endocytosis (i.e., an“endocytosis-competent” host cell) engulfs minicells in accordance withthe invention. Representative endocytosis-competent cells are breastepithelial cells, enterocytes in the gastrointestinal tract, stomachepithelial cells, lung epithelial cells, and urinary tract and bladderepithelial cells.

When effecting delivery to an engulfing-competent mammalian cell withoutthe use of a targeting ligand, the nature of the applicationcontemplated will influence the choice of bacterial source for theminicells employed. For example, Salmonella, Escherichia and Shigellaspecies carry adhesins that are recognized by endocytosis-mediatingreceptors on enterocytes in the gastrointestinal tract, and can besuitable to deliver a drug that is effective for colon cancer cells.Similarly, minicells derived from Helicobacter pylori, carrying adhesinsspecific for stomach epithelial cells, could be suited for deliveryaimed at stomach cancer cells. Inhalation or insufflation can be idealfor administering intact minicells derived from a Pseudomonas speciesthat carry adhesins recognized by receptors on lung epithelial cells.Minicells derived from Lactobacilli bacteria, which carry adhesinsspecific for urinary tract and bladder epithelial cells, could bewell-suited for intraurethral delivery of a drug to a urinary tract or abladder cancer.

Formulations

In one aspect, there is provided a composition comprising (a) aplurality of intact minicells, each minicell of the pluralityencompassing plasmid-free functional nucleic acid, and (b) apharmaceutically acceptable carrier therefor.

The formulation optionally comprises a drug. In one example, theminicell of the formulation contains the drug, while in another theminicell can contain a nucleic acid molecule, such as a plasmid, thatencodes the drug.

The formulations also optionally contain a bispecific ligand fortargeting the minicell to a target cell. The minicell and ligand can beany of those described herein. Thus, the minicell contains a nucleicacid encoding a functional nucleic acid and the bispecific ligandpreferably is capable of binding to a surface component of the minicelland to a surface component of a target mammalian cell.

Formulations can be presented in unit dosage form, e.g., in ampules orvials, or in multi-dose containers, with or without an addedpreservative. The formulation can be a solution, a suspension, or anemulsion in oily or aqueous vehicles, and can contain formulatoryagents, such as suspending, stabilizing and/or dispersing agents. Asuitable solution is isotonic with the blood of the recipient and isillustrated by saline, Ringer's solution, and dextrose solution.Alternatively, formulations can be in lyophilized powder form, forreconstitution with a suitable vehicle, e.g., sterile, pyrogen-freewater or physiological saline. The formulations also can be in the formof a depot preparation. Such long-acting formulations can beadministered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection.

Administration Routes

Formulations described herein can be administered via various routes andto various sites in a mammalian body, to achieve the therapeuticeffect(s) desired, either locally or systemically. Delivery can beaccomplished, for example, by oral administration, by application of theformulation to a body cavity, by inhalation or insufflation, or byparenteral, intramuscular, intravenous, intraportal, intrahepatic,peritoneal, subcutaneous, intratumoral, or intradermal administration.The mode and site of administration is dependent on the location of thetarget cells. For example, cystic-fibrotic cells can be efficientlytargeted by inhaled delivery of the targeted minicells. Similarly, tumormetastasis can be more efficiently treated via intravenous delivery oftargeted minicells. Primary ovarian cancer can be treated viaintraperitoneal delivery of targeted minicells.

Purity

In one aspect, minicells are substantially free from contaminatingparent bacterial cells. Thus, minicell-containing formulationspreferably contain fewer than about 1 contaminating parent bacterialcell per 10⁷ minicells, more preferably contain fewer than about 1contaminating parent bacterial cell per 10⁸ minicells, even morepreferably contain fewer than about 1 contaminating parent bacterialcell per 10⁹ minicells, still more preferably contain fewer than about 1contaminating parent bacterial cell per 10¹⁰ minicells and mostpreferably contain fewer than about 1 contaminating parent bacterialcell per 10¹¹ minicells.

Methods of purifying minicells are known in the art and described ininternational publication number WO03/033519. One such method combinescross-flow filtration (feed flow is parallel to a membrane surface;Forbes, 1987) and dead-end filtration (feed flow is perpendicular to themembrane surface). Optionally, the filtration combination can bepreceded by a differential centrifugation, at low centrifugal force, toremove some portion of the bacterial cells and thereby enrich thesupernatant for minicells.

Another purification method employs density gradient centrifugation in abiologically compatible medium. After centrifugation, a minicell band iscollected from the gradient, and, optionally, the minicells aresubjected to further rounds of density gradient centrifugation tomaximize purity. The method can further include a preliminary step ofperforming differential centrifugation on the minicell-containingsample. When performed at low centrifugal force, differentialcentrifugation will remove some portion of parent bacterial cells,thereby enriching the supernatant for minicells.

Particularly effective purification methods exploit bacterialfilamentation to increase minicell purity. Thus a minicell purificationmethod can include the steps of (a) subjecting a sample containingminicells to a condition that induces parent bacterial cells to adopt afilamentous form, followed by (b) filtering the sample to obtain apurified minicell preparation.

Known minicell purification methods also can be combined. One highlyeffective combination of methods is as follows:

Step A: Differential centrifugation of a minicell producing bacterialcell culture. This step, which can be performed at 2000 g for about 20minutes, removes most parent bacterial cells, while leaving minicells inthe supernatant.

Step B: Density gradient centrifugation using an isotonic and non-toxicdensity gradient medium. This step separates minicells from manycontaminants, including parent bacterial cells, with minimal loss ofminicells. Preferably, this step is repeated within a purificationmethod.

Step C: Cross-flow filtration through a 0.45 μm filter to further reduceparent bacterial cell contamination.

Step D: Stress-induced filamentation of residual parent bacterial cells.This can be accomplished by subjecting the minicell suspension to any ofseveral stress-inducing environmental conditions.

Step E: Antibiotic treatment to kill parent bacterial cells.

Step F: Cross-flow filtration to remove small contaminants, such asmembrane blebs, membrane fragments, bacterial debris, nucleic acids,media components and so forth, and to concentrate the minicells. A 0.2μm filter can be employed to separate minicells from small contaminants,and a 0.1 μm filter can be employed to concentrate minicells.

Step G: Dead-end filtration to eliminate filamentous dead bacterialcells. A 0.45 um filter can be employed for this step.

Step H: Removal of endotoxin from the minicell preparation. Anti-Lipid Acoated magnetic beads can be employed for this step.

Administration Schedules

In general, the formulations disclosed herein can be used at appropriatedosages defined by routine testing, to obtain optimal physiologicaleffect, while minimizing any potential toxicity. The dosage regimen canbe selected in accordance with a variety of factors including age,weight, sex, medical condition of the patient; the severity of thecondition to be treated, the route of administration, and the renal andhepatic function of the patient.

Optimal precision in achieving concentrations of minicell and drugwithin the range that yields maximum efficacy with minimal side effectscan require a regimen based on the kinetics of the functional nucleicacid and drug availability to target sites and target cells.Distribution, equilibrium, and elimination of a minicell or drug can beconsidered when determining the optimal concentration for a treatmentregimen. The dosages of the minicells and drugs can be adjusted whenused in combination, to achieve desired effects.

Moreover, the dosage administration of the formulations can be optimizedusing a pharmacokinetic/pharmacodynamic modeling system. For example,one or more dosage regimens can be chosen and apharmacokinetic/pharmacodynamic model can be used to determine thepharmacokinetic/pharmacodynamic profile of one or more dosage regimens.Next, one of the dosage regimens for administration can be selectedwhich achieves the desired pharmacokinetic/pharmacodynamic responsebased on the particular pharmacokinetic/pharmacodynamic profile. See,e.g., WO 00/67776. In this regard, a dosage regimen for any indicationcan be determined using the approach and model described herein atExample 6, modified for the particular cell of interest.

Specifically, the formulations can be administered at least once a weekover the course of several weeks. In one embodiment, the formulationsare administered at least once a week over several weeks to severalmonths.

More specifically, the formulations can be administered at least once aday for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days.Alternatively, the formulations can be administered about once everyday, about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days ormore.

The formulations can alternatively be administered about once everyweek, about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 weeks or more. Alternatively, the formulations canbe administered at least once a week for about 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks or more.

Alternatively, the formulations can be administered about once everymonth, about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months ormore.

The formulations can be administered in a single daily dose, or thetotal daily dosage can be administered in divided doses of two, three,or four times daily.

In method in which minicells are administered before a drug,administration of the drug can occur anytime from several minutes toseveral hours after administration of the minicells. The drug canalternatively be administered anytime from several hours to severaldays, possibly several weeks up to several months after the minicells.

More specifically, the functional nucleic acid-packaged minicells can beadministered at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before the drug.Moreover, the minicells can be administered at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30 or 31 days before the administration of thedrug. In yet another embodiment, the minicells can be administered atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 weeks or more before the drug. In a further embodiment, theminicells can be administered at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or 12 months before the drug.

In another embodiment, the minicell is administered after the drug. Theadministration of the minicell can occur anytime from several minutes toseveral hours after the administration of the drug. The minicell canalternatively be administered anytime from several hours to severaldays, possibly several weeks up to several months after the drug.

The following examples are illustrative only, rather than limiting, andprovide a more complete understanding of the invention.

EXAMPLES 1. Direct Packaging of Regulatory RNA into Intact Minicells InVitro

Bacterially derived intact minicells were prepared and purified asdescribed in U.S. Published Application No. US 2004/0265994. Cy3-labeledglyceraldehydes-3-phosphate dehydrogenase (GAPDH) siRNA was obtained(excitation max (λ_(max)) 547 nm, emission max (λ_(max)) 563 nm),product of Ambion (Austin, Tex. USA), and was reconstituted innuclease-free water to a final concentration of 50 μM.

Approximately 10⁷ minicells were resuspended in 1× Phosphate BufferSolution (PBS) (Gibco) and were co-incubated with 1 μM of Cy3-labeledGAPDH siRNA. The incubation was carried out for 2 hours at 37° C., withgentle mixing. Control minicells were mock loaded by incubation with1×PBS alone. Post loading minicells were pelleted and washed twice with1×PBS by centrifugation for 10 minutes at 16,200×g. Experimental andcontrol minicells were observed under a fluorescence DMLB microscope,product of Leica (Germany) with attached D70 camera, product of OlympusMicroscopes (Germany). Images were acquired using 100× oil immersionlens.

The above co-incubation experiments were also carried out underdifferent experimental conditions such as incubation at roomtemperature, 37° C., and 4° C. Additionally, the co-incubation timeswere varied to include 1 hour, 2 hours, 4 hours, and 12 hours,respectively.

As shown in FIG. 1B, the intact the siRNA molecules rapidly diffusedinto the minicells. The 2-hour incubation at 37° C. was sufficient toachieve highly significant packaging of the minicells.

In order to determine whether the siRNA molecules were inside theminicells or adherent on minicell surfaces, the minicells withCy3-fluorescence-labeled siRNA were incubated with exonucleasesovernight, followed by a repeat of fluorescence microscopy. The resultswere identical to those shown in FIG. 1B, indicating that the siRNAswere internalized by the minicells and were not adherent on the minicellsurface.

2. In Vitro Transfection of Human Breast Cancer Cells with RegulatoryRNA-Packaged, Bispecific Antibody-Targeted Minicells

To demonstrate that minicells carrying regulatory RNA are stable inserum in-vitro and that they can be internalized byspecifically-targeted mammalian cells, the following experiment wascarried out.

siRNA directed to polo-kinase 1 (Plk1) was synthesized with a targetsequence 5′-GGTGGATGTGTGGTCCATTTT-3′, and tagged with a fluorescent tag,AlexaFluor 488. Polio kinases have multiple functions during the entryinto mitosis, centrosome maturation, bipolar spindle formation, thesegregation of chromosomes and cytokinesis, and, crucially, the fidelitymonitoring of checkpoint control (Glover et al., 1998; Barr et al.,2004; van de Weerdt and Medema 2006). In humans, Plk1 is the bestcharacterized member of this family. Plk1 is associated withtumorigenesis and belongs to the family of serine/threonine kinases,which represent attractive targets for novel chemotherapeutics.Accordingly, Plk1 is deemed a promising target for anticancer drugdevelopment (Strebhardt and Ullrich, 2006).

Minicells were purified, and 10⁹ minicells were packaged withanti-^(AF488)Plk1 siRNA, as described in Example 1. A bispecificantibody (BsAb) was prepared, carrying anti-S. typhimurium O-antigen andanti-human EGFR specificities, and was attached to theminicells_(AF488-Plk1-siRNA) as described in PCT published applicationWO 05/056749. The resultant minicells were designated^(EGFR)minicells_(AF488-Plk1-siRNA). These minicells (10⁹) wereincubated with human breast cancer cells, in tissue culture, at adensity of 10,000 minicells:1 tumor cell. The incubation was carried outfor 1 hour, 2 hours, 4 hours and 24 hours. At each time point, the cellswere collected and stained with DAPI (nuclear stain, blue fluorescence).The cells were observed using the IX81 confocal microscope (Olympus) andthe CellR software.

By 1 hour the fluorescent, siRNA-carrying minicells had adhered to theMDA-MB-468 cells (see FIG. 2). This attachment was due, it is believed,to the binding to the minicell-attached BsAb, which targeted the EGFreceptor on the MDA-MB-468 cells, since the control incubation withnon-targeted minicells_(AF488-Plk1-siRNA) were washed away and did notshow any green fluorescence associated with the MDA-MB-468 cells. By 2hours post-incubation, the ^(EGFR)minicells_(AF488-Plk1-siRNA) wereinternalized within the MDA-MB-468 cells and exhibited intense greenfluorescence. By 24 hours most of the green fluorescence was gone,indicating that the internalized minicells had been broken down,presumably within the phagolysosomes.

3. Extraction and Quantification of siRNA from Intact Minicells

Since siRNAs do not occur naturally in bacterial cells orbacterially-derived minicells, it is unsurprising that no establishedmethodology exists for extracting siRNA from such particles.Accordingly, the present inventors developed a method for quantitativeextraction of siRNAs that are packaged in intact minicells, pursuant tothe invention.

Kinesin spindle protein (KSP), also known as “kinesin-5” and “Eg5,” is amicrotubule motor protein. It is essential to the formation of bipolarspindles and to proper segregation of sister chromatids during mitosis(Enos and Morris, 1990; Blangy et al., 1995; Sawin and Mitchison, 1995;Dagenbach and Endow, 2004). Inhibition of KSP causes the formation ofmonopolar mitotic spindles, activates the spindle assembly checkpoint,and arrests cells at mitosis, leading to subsequent cell death (Blangyet al., 1995, Caner et al., 1999; Kapoor et al., 2000; Tao et al.,2005).

An siRNA against KSP was selected for packaging in minicells, to informthe optimization of siRNA extraction from minicells in accordance withthe invention. More specifically, KSP-1-siRNA double-strandedoligonucleotide sequences (sense strand; 5′-AAC TGG ATC GTA AGA AGGCAG-3′) were synthesized and packaged into minicells, pursuant to theprocedures set out in Example 1, supra.

Minicells_(siRNA-KSP) (10¹⁰) and a comparable number of control emptyminicells were processed, using a number of commercially availablenucleic acid extraction kits. The results showed that the mirVana miRNAisolation kit (Ambion) provided quantitative extraction of the siRNA-KSPfrom intact minicells. The procedure was carried out according to themanufacturer's instructions.

The purified siRNA first was stained with an ultra-sensitive fluorescentnucleic acid dye, RiboGreen™, product of Molecular Probes Inc. (Eugene,Oreg. USA), followed by quantitation using the NanoDrop ND-3300Fluorospectrometer, product of NanoDrop Technologies Inc. (Wilmington,Del. USA), also according to the manufacturer's instructions. RNA-boundRiboGreen™ has an excitation maximum of ˜500 nm and an emission maximumof ˜525 nm.

The results showed that the minicells were able to carry the siRNAs.10¹⁰ empty minicells carried ˜1.4 μg RNA, presumably a background levelof endogenously formed bacterial RNA. The same number ofminicells_(siRNA-KSP) carried ˜2.7 μg RNA, comprising endogenousbacterial RNA plus exogenously packaged siRNA-KSP Thus, these datademonstrate that 10¹⁰ minicells can package at least ˜1.3 μg ofexogenously packaged siRNA.

4. In Vivo Demonstration of Anti-Tumor Effects Achieved by RegulatoryRNA-Packaged Minicells

The following studies were conducted to show that regulatoryRNA-packaged minicells could deliver intact, regulatory RNA intherapeutically effective concentrations to tumor cells in vivo.

siRNA against KSP, as described in Examiner 3, was selected to packagein the minicells according to the present invention. Minicells werepurified, and 10⁹ minicells were packaged with anti-KSP siRNA, asdescribed in Example 1. A BsAb also was prepared and attached to theminicells_(siRNA-KSP), as described in Example 2, to generate^(EGFR)minicells_(siRNA-KSP).

The mice used in this example were purchased from Animal ResourcesCentre (Perth, Wash., Australia), and all animal experiments wereperformed in compliance with the guide of care and use of laboratoryanimals, with Animal Ethics Committee approval. The experiments wereperformed in the NSW Agriculture-accredited small animal facility atEnGeneIC Pty Ltd (Sydney, New South Wales, Australia).

Human breast cancer cells (MDA-MB-468, ATCC) were grown in tissueculture in RPMI 1640 medium supplemented with GIBCO-BRL 5% Bovine CalfSerum, product of Invitrogen Corporation (Carlsbad, Calif. USA), andglutamine (Invitrogen) in a humidified atmosphere of 95% air and 5% CO₂at 37° C. 1×10⁶ cells in 50 μl serum-free media were mixed together with50 μl growth factor reduced matrigel, product of BD Biosciences(Franklin Lakes, N.J. USA). By means of a 23-gauge needle, the cellswere injected subcutaneously between the shoulder blades of each mouse.The tumors were measured twice a week, using an electronic digitalcaliper (precision to 0.001), product of Mitutoyo (Japan), and meantumor volume was calculated using the formula: length (mm)×width²(mm)×0.5=volume (mm³).

The various treatments commenced once the tumors reached volumes between170 mm³ and 200 mm³, and mice were randomized to two different groups ofeight per group. Control group 1 received sterile saline, whileexperimental group 2 received ^(EGFR)minicells_(siRNA-KSP). (10⁹), fourtimes per week.

As shown in FIG. 3, ^(EGFR)minicells_(siRNA-KSP) provided a highlysignificant anti-tumor effect compared to the saline controls. Theresults demonstrate that (a) siRNAs were stable within the minicells invivo, (b) intact and fully functional siRNAs were delivered to the tumorcells in vivo, and (c) the minicell delivered therapeuticallysignificant concentrations of siRNA to the tumor cells in vivo.

5. Demonstration of Tailor-Made Cancer Therapy Via Treatment withRegulatory RNA-Packaged Minicells, Followed by Drug-Packaged Minicells

Most anti-cancer therapies are associated with drug resistance. The sameis true for regulatory RNA treatments, since genetic mutations in tumorcells can render the regulatory RNA ineffective if the target genemutates within the sequence targeted by the regulatory RNA.

There has been no effective strategy to address drug resistance incancer patients. Instead, new drugs must be administered to bypass themutation. This approach faces serious difficulties, however, since mostanti-cancer drugs are highly toxic, and combined therapies augment thattoxicity, resulting in dose limitation and frequent abandonment oftherapy when the patient can no longer cope with the toxicity. Thefollowing study was conducted to assess the efficacy of regulatoryRNA-packaged minicells in addressing such resistance.

As described above, minicells were purified and packaged (10⁹) withanti-KSP or anti-Plk1 siRNA. Also as previously described, bispecificantibody was prepared, carrying anti-S. typhimurium O-antigen andanti-human EGFR specificities, and was attached to theminicells_(siRNA-KSP) to generate ^(EGFR)minicells_(siRNA-KSP).

Human colon cancer (HCT116; ATCC) xenografts were established in nudemice, as described in Example 3, and were treated i.v. as follows: Group1 mice received sterile saline, and mice of groups 2, 3 and 4 weretreated for the first 10 doses (see FIG. 4) with10^(9 EGFR)minicells_(siRNA-Plk1), ^(EGFR)minicells_(siRNA-KSP-1), and^(EGFR)minicells_(siRNA-KSP-2), respectively. The Plk1 and KSP-1sequences were as shown in the examples above. siRNA-KSP-2 (sensestrand; 5′ CTGAAGACC TGAAGACAAT 3′) targets a different segment of KSPmRNA. After day 33, mice in groups 2, 3 and 4 were treated with twodoses of ^(EGFR)minicells_(carboplatin).

The results showed (FIG. 4) that post-day 26, the tumors were becomingresistant to the siRNA treatments. Accordingly, the mice in groups 2, 3and 4 were treated for the four subsequent doses, with all three doses^(EGFR)minicells_(siRNA-Plk1), +^(EGFR)minicells_(siRNA-KSP-1),+^(EGFR)minicells_(siRNA-KSP-2)) combined in equal quantities, i.e.,˜3×10⁸ of each minicell type. In addition, by day 33 the tumors werehighly resistant to all the siRNAs (FIG. 4). Following administration of^(EGFR)minicells_(carboplatin), the tumor growth in groups 3 and 4 micewas retarded significantly. Following administration of^(EGFR)minicells_(carboplatin), group 3 mice showed a significantregression in tumor volume.

These data show that drug-resistant tumor cells can be treatedeffectively in vivo, pursuant to the present invention. In particular,(1) sequential administrations of targeted minicells, carryingregulatory RNA sequences designed to reduce tumor burden significantly,are followed, when the tumor cells become resistant to thesiRNA-mediated anti-tumor effect, by (2) targeted minicells carrying adrug that does not act on the same protein targeted by the regulatoryRNA.

6. Demonstration of Target Protein Knockdown in Tumor Cells andResultant Arrest in Cell Growth Following Targeted Delivery of aTherapeutically Effective Amount of Regulatory RNA Packaged in IntactMinicells

To demonstrate that the inventive methods package therapeuticallyeffective amounts of regulatory RNA in intact minicells, it wasnecessary to demonstrate that bispecific antibody-targeted, regulatoryRNA-packaged minicells could efficiently and effectively trigger tumorcell-growth arrest and induce apoptotic cell death.

In a humidified atmosphere of 95% air and 5% CO₂ at 37° C., humancolonic epithelial cancer cells (HCT116) were grown in tissue culture inRPMI 1640 medium, supplemented with 5% Bovine Calf Serum and glutamine.As described above, minicells were purified, were packaged with siRNAdirected against Plk1 or KSP, and were attached to a BsAb that carriedanti-S. typhimurium O-antigen and anti-human EGFR specificities. Thus,from the ^(EGFR)minicells_(siRNA-KSP), ^(EGFR)minicells_(siRNA-Plk1),and minicells (control) were generated ^(EGFR)minicells_(siRNA-KSP),^(EGFR)minicells_(siRNA-Plk1) and ^(EGFR)minicells. HCT116 cells wereseeded in six-well plates, and the experimental and control groups weretransfected at a ratio of 5,000 minicells:1 HCT116 cell. An additional,cells-only control was included.

After 2 hours incubation with minicells, the wells were washed threetimes with fresh PBS. Wells were harvested at 4 hours, 8 hours, 16hours, 24 hours, 32 hours and 48 hours post-transfection, and cells werefixed in cold 70% ethanol and incubated at 4° C. for 30 minutes. Thecells were washed twice in phosphate-citrate buffer (pH 7.8) and weretreated with 100 mg/ml of RNAse, to ensure that only the DNA wasstained. The cells were stained with propidium iodide (nucleic acidstain) and then were analyzed using a FACSCalibur™ flow cytometer,product of Becton Dickinson (Franklin Lakes, N.J. USA), at MacquarieUniversity (Sydney, Australia), and the CELL Questacquisition-and-analysis software, also a Becton Dickinson product.

FACS analysis of the cells showed that, by 4 and 8 hourspost-transfection (FIG. 5A), cells treated with either^(EGFR)minicells_(siRNA-KSP) or ^(EGFR)minicells_(siRNA-Plk1) werecharacterized by a robust G2 cell-cycle arrest. Control cells, eithercells only or treated with ^(EGFR)minicells, showed no adverse effects.These cells showed normal G1, S, and G2 phases of the cell cycle. By 16and 24 hours, the experimental cells displayed not only a robust G2arrest but also a large number of apoptotic cells (FIG. 6). By 32 and 48hours, most of the cells in the experimental groups were apoptotic andhad turned into cell debris (See in particular brackets in FIG. 7).

These results demonstrate that the targeted intact minicells werepackaged with a therapeutically effective amount of regulatory RNA, andthat the minicells of the invention were highly efficient andsignificantly effective in target protein knockdown within tumor cells,resulting in apoptotic cell death.

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We claim:
 1. A composition comprising (a) a plurality of intact,bacterially derived minicells, each minicell of said pluralityencompassing regulatory RNA that is packaged in said minicell, and (b) apharmaceutically acceptable carrier therefor, wherein said regulatoryRNA is selected from the group consisting of siRNA, miRNA and shRNA,wherein there is an absence from said minicells of a construct for insitu expression of said regulatory RNA, and wherein said pluralitycontains a therapeutically effective amount of said regulatory RNA. 2.The composition of claim 1, wherein said regulatory RNA is notchemically modified.
 3. The composition of claim 1, wherein saidregulatory RNA specifically interferes with expression of a protein thatcontributes to drug resistance.
 4. The composition of claim 3, whereinsaid protein is P-glycoprotein, MDR-2, or MDR-3.
 5. The composition ofclaim 3, wherein said protein is MRP2, BCR-ABL, STI-571resistance-associated protein, lung resistance-related protein,cyclooxygenase-2, nuclear factor kappa, XRCC1, ERCC1, GSTP1, mutantβ-tubulin, or a growth factor.
 6. The composition of claim 1, furthercomprising a bispecific ligand.
 7. The composition of claim 6, whereinsaid bispecific ligand comprises a first arm that carries specificityfor a minicell surface structure and a second arm that carriesspecificity for a non-phagocytic mammalian cell surface receptor.
 8. Thecomposition of claim 7, wherein said mammalian cell surface receptor iscapable of activating receptor-mediated endocytosis of said minicell. 9.The composition of claim 1, wherein said composition contains fewer thanabout 1 contaminating parent bacterial cell per 10¹⁰ minicells.
 10. Thecomposition of claim 1, wherein said composition contains fewer thanabout 1 contaminating parent bacterial cell per 10¹¹ minicells.
 11. Amethod of delivering regulatory RNA, comprising bringing a compositionof claim 1 into contact with mammalian cells such that said mammaliancells engulf minicells of said plurality, whereby said regulatory RNA isreleased into the cytoplasm of the mammalian cells.
 12. The method ofclaim 11, wherein contact between said minicells and said mammaliancells occurs in vitro.
 13. The method of claim 11, wherein contactbetween said minicells and said mammalian cells occurs in vivo.